Modulators of p38 MAP kinase

ABSTRACT

Compounds of formula I are effective modulators of p38 MAP kinase.  
                 
 
     wherein R 1  is H, lower alkyl, or OR 5 , where R 5  is aryl, heterocyclyl, acyl, N—R 6 -4-piperidinyl, N—R 6 -4-piperidinylmethyl, or N—R 6 -4-pipelidinylethyl, where aryl and heterocyclyl are substituted with 1-3 R 6  substituents selected from the group consisting of H, lower alkyl, lower alkenyl, OH, NO 2 , NH 2 , halo, trihalomethyl, —CN, SH, SO, SO 2 , SO 3 H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is lower alkyl; R 2  is H or lower alkyl; R 3  is aryl substituted with 1-3 R 6  substituents; R 4  is H, lower alkyl, aryl, aralkyl, —OH, —NH 2 , —NHR, or —OR, where R is lower alkyl or aryl-lower alkyl; and A is —CH— or —N—; and pharmaceutically acceptable salts thereof.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to provisional patent applicationSer. No. 60/184,368, filed Feb. 23, 2000, from which priority is claimedunder 35 USC §119(e)(1) and which is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to the fields of molecularbiology and pharmaceutical methods of treatment. More particularly, theinvention relates to compounds, compositions, and methods for modulatingp38 MAP kinase activity.

BACKGROUND OF THE INVENTION

[0003] Activation of members of the mitogen-activated protein kinase(MAPK) family represents one of the major mechanisms used by eukaryoticcells to transduce extracellular signals into cellular response (J.Blenis, Proc Natl Acad Sci USA (1993) 90:5889). One member of thiskinase family, p38, has been implicated in signaling pathways used bybiologically important stimuli including products of microbialpathogens, cytokines, UV light and increased extracellular osmolarity.Most recently p38 activation was found to be correlated with apoptosisin neuronal cells following withdrawal of nerve growth factor. Thekinase p38 is activated by a subset of the known dual-specificity MAPKkinases (MEKs or MKKs); low molecular weight GTP-binding proteins RAC1and Cdc42 have also been shown to play a role in regulating p38activation by some stimuli. A complete understanding of how p38activation regulates cellular responses requires identification ofspecific substrates for this enzyme.

SUMMARY OF THE INVENTION

[0004] We have now identified compounds that modulate the activity ofp38.

[0005] One aspect of the invention is a compound of formula 1:

[0006] wherein R₁ is H, lower alkyl, or —OR₅, where R₅ is aryl,heterocyclyl, acyl, N—R₆-4-piperidinyl, N—R₆-4-piperidinylmethyl, orN—R₆-4-piperidinylethyl, where aryl and heterocyclyl are substitutedwith 1-3 R₆ substituents selected from the group consisting of H, loweralkyl, lower alkenyl, OH, NO₂, NH₂, halo, trihalomethyl, —CN, SH, SO,SO₂, SO₃H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is loweralkyl; R₂ is H or lower alkyl; R₃ is aryl substituted with 1-3 R₆substituents as set forth above; R₄ is H, lower alkyl, aryl, aralkyl,—OH, —NH₂, —NHR, or —OR, where R is lower alkyl or aryl-lower alkyl; andA is —CH— or —N—; and pharmaceutically acceptable salts thereof.

[0007] Another aspect of the invention is a pharmaceutical composition,comprising a compound of formula 1 and a pharmaceutically acceptableexcipient.

[0008] Another aspect of the invention is a method for modulating p38activity in a cell, comprising administering an effective amount of acompound of formula 1 to said cell.

DETAILED DESCRIPTION

[0009] Definitions:

[0010] “Compound of formula 1” refers to compounds having the structure

[0011] wherein R₁ is H, lower alkyl, or OR₅, where R₅ is aryl,heterocyclyl, acyl, N—R₆-4-piperidinyl, N—R₆-4-piperidinylmethyl, orN—R₆-4-piperidinylethyl, where aryl and heterocyclyl are substitutedwith 1-3 R₆ substituents selected from the group consisting of H, loweralkyl, lower alkenyl, OH, NO₂, NH₂, halo, trihalomethyl, —CN, SH, SO,SO₂, SO₃H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is loweralkyl; R₂ is H or lower alkyl; R₃ is aryl substituted with 1-3 R₆substituents as set forth above; R₄ is H, lower alkyl, aryl, aralkyl,—OH, —NH₂, —NHR, or —OR, where R is lower alkyl or aryl-lower alkyl; andA is —CH— or —N—; and pharmaceutically acceptable salts and estersthereof. It should be noted that the two amidine nitrogen atoms cantautomerize if R₂ is H, in which case either R₁ or R₃ can be bound tothe imide nitrogen (the nitrogen double-bonded to carbon). Thus, noabsolute geometry is indicated or intended around the R₁—N═C bond.

[0012] The term “lower alkyl” refers to radicals containing carbon andhydrogen, without unsaturation, having from one to six carbon atoms,inclusive. Lower alkyl radicals can be straight or branched. Exemplarylower alkyl radicals include, without limitation, methyl, ethyl, propyl,isopropyl, hexyl, t-butyl, and the like. The term “lower alkenyl” refersto a hydrocarbon radical having 2-6 carbon atoms, and at least onedouble bond. Exemplary lower alkenyl radicals include, withoutlimitation, vinyl, propenyl, butenyl, and the like.

[0013] The term “aryl” refers to an aromatic carbocyclic or heterocyclicmoiety, having one, two, or three rings. Exemplary aryl radicalsinclude, without limitation, phenyl, naphthyl, pyridyl, pyrimidyl,triazyl, quinazolinyl, pyranyl, thiazolyl, and the like. The terms“aralkyl” and “aryl-lower alkyl” refer to an aryl moiety joined to alower alkyl moiety, for example benzyl, phenethyl, 2-phenylpropyl,naphthylmethyl, and the like.

[0014] The term “halo” refers to fluoro, chloro, bromo, and iodo.

[0015] The term “leaving group” refers to a radical that is easilydisplaced by a nucleophile in an S_(N)2 displacement reaction. Exemplaryleaving groups include, without limitation, sulfonates such as tosylateand mesylate, silanes such as t-butyl-dimethylsilane, halogens such asbromo and chloro, and the like.

[0016] The term “pharmaceutically acceptable salts and esters” refers toderivatives of compounds of formula I obtained by addition of an acid orbase to the compound, or condensation with an alcohol or carboxylic acidto form an ester. In either case, the acid, base, alcohol, or carboxylicacid must not be unacceptably toxic at the concentrations at which thecompound is administered. Suitable acids include, without limitation,inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid or phosphoric acid; organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, malonic acid, succinicacid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid and the like.

[0017] The term “modulate” as used herein refers to an alteration in p38MAP kinase activity, and includes both increases and decreases inactivity. Modulation of activity can occur as the result of directinteraction of a compound with p38, interaction with another compound orprotein that affects p38 activity directly or indirectly, or by alteringthe expression of p38 or of a protein that interacts with p38 directlyor indirectly.

[0018] General Method:

[0019] Compounds of the invention are prepared by any suitable syntheticscheme. For example, an intermediate of formula A can be treated with analcohol (R-OH) and NaH or Na—OR to form intermediate B.

[0020] Intermediate B can then be treated with an intermediate of theform R₁—L (where L is a leaving group) in the presence of a strong,hindered base to provide an intermediate of formula C. This intermediateis then treated with an amine of the formula HNR₂R₃ under acidic orbasic catalysis to provide the compound of formula 1.

[0021] Compounds of the invention can be tested for activity by anyavailable assay suitable for determining p38 activity or interferencetherewith.

[0022] Compounds of the invention can be administered to a subject, orcan be applied directly to cells, for example in a cell culture. Ifadministered to a cell culture, the compound is preferably firstsuspended or dissolved in a suitable carrier. Suitable carriers include,without limitation, water, saline solution, dimethylsulfoxide (DMSO) andsolutions thereof, cell culture media, and the like.

[0023] Useful pharmaceutical carriers for the preparation of thepharmaceutical compositions hereof can be solids or liquids. Thus, thecompositions can take the form of tablets, pills, capsules, powders,sustained release formulations, solutions, suspensions, elixirs,aerosols, and the like. Carriers can be selected from the various oils,including those of petroleum, animal, vegetable or synthetic origin, forexample, peanut oil, soybean oil, mineral oil, sesame oil, and the like.Water, saline, aqueous dextrose, and glycols are preferred liquidcarriers, particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, cellulose, talc, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate,sodium stearate, glycerol monostearate, sodium chloride, dried skimmilk, glycerol, propylene glycol, water, ethanol, and the like. Othersuitable pharmaceutical carriers and their formulations are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

[0024] A compound of formula I or a pharmaceutical compositioncontaining same is administered via any of the usual and acceptablemethods known in the art, either singly or in combination with anothercompound or compounds of the present invention or other pharmaceuticalagents. These compounds or compositions can thus be administered orally,systemically (e.g., transdermally, intranasally or by suppository) orparenterally (e.g., intramuscularly, subcutaneously and intravenously),and can be administered either in the form of solid or liquid dosagesincluding tablets, solutions, suspensions, aerosols, and the like, asdiscussed in more detail above. It is preferred to administer compoundsof formula I orally. The formulation can be administered in a singleunit dosage form for continuous treatment or in a single unit dosageform ad libitum when relief of symptoms is specifically required.

EXAMPLES

[0025] The following examples are provided as a guide for thepractitioner of ordinary skill in the art. Nothing in the examples isintended to limit the claimed invention. Unless otherwise specified, allreagents are used in accordance with the manufacturer's recommendations,and all reactions are performed at ambient temperature and pressure.

Example 1 (p38 Assay)

[0026] (A) Protocol: Recombinant p38 was pre-incubated for 30 minutes atroom temperature in ADB (Assay Dilution Buffer: 20 mM MOPS, pH 7.2, 20mM MgCl₂, 5 mM EGTA, 1 mM DTT, 25 mM β-glycerolphosphate, 2 mM pNPP, 0.1mM sodium orthovanadate) with recombinant GST-ATF-2 and compounds atvarious concentrations. The DMSO concentration was held constant at 0.1%(v/v) independent of the compound concentration. Kinase reactions werestarted with a mixture of ³²P-γ-ATP and ATP in ADB and were incubatedfor 30 minutes at 37° C. Final concentrations in kinase reactions was0.625 ng/μl recombinant p38 (Biomol, catalog number SE-150), 62.5 ng/μlrecombinant GST-ATF-2 (Upstate Biotechnology, catalog #12-367) and 30 μMATP (Amersham, catalog #AA0068, used within two weeks of the referencedate). Reactions were terminated by the addition of stopmix (finalconcentrations: 62.5 mM Tris pH 6.8, 10% (v/v) glycerol, 5% (v/v)B-mercaptoethanol, 20 mg/ml SDS, 1 mg/ml bromophenol blue). At thispoint samples were either processed directly or stored at 4° C. Foranalysis, samples were heated to 95° C. for 5 min and proteins wereseparated by SDS-PAGE using 8-16% precast gels (Novex). Followingelectrophoresis, gels were stained in 0.25% (w/v) brilliant blue R, 40%(v/v) methanol and 7% (v/v) acetic acid to visualize protein bands. Gelswere then incubated with 2% glycerol and exposed overnight toPhospholmager screens (Molecular Dynamics). Screens were scanned using aPhospholmager SI (Molecular Dynamics) and the amount of radioactive ³²Pincorporated into GST-ATF-2 was quantified using ImageQuant (MolecularDynamics) software. Data were normalized against control samples treatedwith DMSO and were plotted as percent of p38 activity of samples ascompared to control. IC₅₀ values were estimated by estimating from thegraph the concentration of each compound at which p38 activity reached50% of the control.

[0027] (B) Results: In the above-described assay, two tested compounds(X and Y below) demonstrated activity.

[0028] N-(3-trifluoromethylphenyl)-N′-isopropyl-4-amidinopyridine (X)exhibited an IC₅₀ of 0.75 μM, whileN-(4-chloromethylphenyl)-N′-isopropyl-4-amidinopyridine (Y) exhibited anIC₅₀ of 4.0 μM. In growth rescue ARC experiments performed in yeast(following the methods taught in U.S. Ser. No. 09/187,918, WO/99/24563,both incorporated herein by reference in full), the compoundsdemonstrated an in vivo IC₅₀ of 13.2 μM and 546.2 μM, respectively.

What is claimed:
 1. A compound of formula 1:

wherein R₁ is H, lower alkyl, or OR₅, where R₅ is aryl, heterocyclyl, acyl, N—R₆-4-piperidinyl, N—R₆-4-piperidinylmethyl, or N—R₆-4-piperidinylethyl, where aryl and heterocyclyl are substituted with 1-3 R₆ substituents selected from the group consisting of H, lower alkyl, lower alkenyl, OH, NO₂, NH₂, halo, trihalomethyl, —CN, SH, SO, SO₂, SO₃H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is lower alkyl; R₂ is H or lower alkyl; R₃ is aryl substituted with 1-3 R₆ substituents; R₄ is H, lower alkyl, aryl, aralkyl, —OH, —NH₂, —NHR, or —OR, where R is lower alkyl or aryl-lower alkyl; and A is —CH— or —N—; and pharmaceutically acceptable salts thereof.
 2. The compound of claim 1 ), wherein R₄ is H and A is —CH—.
 3. The compound of claim 2 ), wherein R₁ is lower alkyl and R₂ is H.
 4. The compound of claim 3 ), wherein R₃ is —(C₆H₂)R₉R₈R₇, wherein R₉, R₈, and R₇ are each independently H, lower alkyl, lower alkyl substituted with one or more halo, lower alkenyl, OH, NO₂, NH₂, halo, trihalomethyl, —CN, SH, SO, SO₂, SO₃H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is lower alkyl.
 5. The compound of claim 4 ), wherein R₁ is isopropyl, R₉ is 3-trifluoromethyl, and R₈ and R₇ are each H.
 6. The compound of claim 1 ), wherein A is —N— and R₄ is —NHR₆.
 7. A method for modulating p38 activity, comprising: Administering to a subject cell that expresses p38 an effective amount of a compound of formula 1:

wherein R₁ is H, lower alkyl, or OR₅, where R₅ is aryl, heterocyclyl, acyl, N—R₆-4-piperidinyl, N—R₆-4-piperidinylmethyl, or N—R₆-4-piperidinylethyl, where aryl and heterocyclyl are substituted with 1-3 R₆ substituents selected from the group consisting of H, lower alkyl, lower alkenyl, OH, NO₂, NH₂, halo, trihalomethyl, —CN, SH, SO, SO₂, SO₃H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is lower alkyl; R₂ is H or lower alkyl; R₃ is aryl substituted with 1-3 R₆ substituents; R₄ is H, lower alkyl, aryl, aralkyl, —OH, —NH₂, —NHR, or —OR, where R is lower alkyl or aryl-lower alkyl; and A is —CH— or —N—; and pharmaceutically acceptable salts thereof.
 8. The method of claim 7 ), wherein said subject cell comprises a mammalian cell in a cell culture.
 9. The method of claim 7 ), wherein said subject cell comprises a cell within an organism.
 10. A pharmaceutical composition, comprising: A compound of formula 1,

wherein R₁ is H, lower alkyl, or OR₅, where R₅ is aryl, heterocyclyl, acyl, N—R₆-4-piperidinyl, N—R₆-4-piperidinylmethyl, or N—R₆-4-piperidinylethyl, where aryl and heterocyclyl are substituted with 1-3 R₆ substituents selected from the group consisting of H, lower alkyl, lower alkenyl, OH, NO₂, NH₂, halo, trihalomethyl, —CN, SH, SO, SO₂, SO₃H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is lower alkyl; R₂ is H or lower alkyl; R₃ is aryl substituted with 1-3 R₆ substituents; R₄ is H, lower alkyl, aryl, aralkyl, —OH, —NH₂, —NHR, or —OR, where R is lower alkyl or aryl-lower alkyl; and A is —CH— or —N—; and pharmaceutically acceptable salts thereof; and A pharmaceutically acceptable carrier.
 11. The composition of claim 10 ), wherein R₄ is H and A is —CH—.
 12. The compound of claim 11 ), wherein R₁ is lower alkyl and R₂ is H.
 13. The compound of claim 12 ), wherein R₃ is —(C₆H₂)R₉R₈R₇, wherein R₉, R₈, and R₇ are each independently H, lower alkyl, lower alkyl substituted with one or more halo, lower alkenyl, OH, NO₂, NH₂, halo, SH, SO, SO₂, SO₃H, —OR, —COR, —COOR, —CONHR, —OCOR, and —NCOR, where R is lower alkyl.
 14. The compound of claim 13 ), wherein R₁ is isopropyl, R₅ is 3-trifluoromethyl, and R₆ and R₇ are each H. 